Asymmetric benzoxanthene dyes

ABSTRACT

A class of asymmetric monobenzoxanthene compounds useful as fluorescent dyes are disclosed having the structure ##STR1## wherein Y 1  and Y 2  are individually hydroxyl amino, imminium, or oxygen, R 1  -R 8  are hydrogen, fluorine, chlorine, alkyl, alkene, alkyne, sulfonate, amino, amido, nitrile, alkoxy, linking group, and combinations thereof, and R 9  is acetylene, alkane, alkene, cyano, substituted phenyl, and combinations thereof. The invention further includes novel intermediate compounds useful for the synthesis of asymmetric benzoxanthene compounds having the general structure ##STR2## where substituents R 3  -R 7  correspond to like-referenced substituents in the structure of described above, and Y 2  is hydroxyl or amine. In another aspect, the invention includes methods for synthesizing the above dye compounds and intermediates. In yet another aspect, the present invention includes reagents labeled with the asymmetric benzoxanthene dye compounds, including deoxynucleotides, dideoxynucleotides, phosphoramidites, and polynucleotides. In an additional aspect, the invention includes methods utilizing such dye compounds and reagents including dideoxy polynucleotide sequencing and fragment analysis methods.

FIELD OF THE INVENTION

This invention relates generally to fluorescent dye compounds useful asmolecular probes. More specifically, this invention relates toasymmetric benzoxanthene dyes useful as fluorescent labeling reagents.

BACKGROUND

The non-radioactive detection of biological analytes is an importanttechnology in modem analytical biotechnology. By eliminating the needfor radioactive labels, safety is enhanced and the environmental impactof reagent disposal is greatly reduced, resulting in decreased costs foranalysis. Examples of methods utilizing such non-radioactive detectionmethods include DNA sequencing, oligonucleotide probe methods, detectionof polymerase-chain-reaction products, immunoassays, and the like.

In many applications the independent detection of multiple spatiallyoverlapping analytes in a mixture is required, e.g., single-tubemultiplex DNA probe assays, immuno assays, multicolor DNA sequencingmethods, and the like. In the case of multi-loci DNA probe assays, byproviding multicolor detection, the number of reaction tubes may bereduced thereby simplifying the experimental protocols and facilitatingthe manufacturing of application-specific kits. In the case of automatedDNA sequencing, multicolor labeling allows for the analysis of all fourbases in a single lane thereby increasing throughput over single-colormethods and eliminating uncertainties associated with inter-laneelectrophoretic mobility variations.

Multiplex detection imposes a number of severe constraints on theselection of dye labels, particularly for analyses requiring anelectrophoretic separation and treatment with enzymes, e.g., DNAsequencing. First, it is difficult to find a collection of dyes whoseemission spectra are spectrally resolved, since the typical emissionband half-width for organic fluorescent dyes is about 40-80 nanometers(nm) and the width of the available spectrum is limited by theexcitation light source. As used herein the term "spectral resolution"in reference to a set of dyes means that the fluorescent emission bandsof the dyes are sufficiently distinct, i.e., sufficientlynon-overlapping, that reagents to which the respective dyes areattached, e.g. polynucleotides, can be distinguished on the basis of thefluorescent signal generated by the respective dyes using standardphotodetection systems, e.g. employing a system of band pass filters andphotomultiplier tubes, charged-coupled devices and spectrographs, or thelike, as exemplified by the systems described in U.S. Pat. Nos.4,230,558, 4,811,218, or in Wheeless et al, pgs. 21-76, in FlowCytometry: Instrumentation and Data Analysis (Academic Press, New York,1985). Second, even if dyes with non-overlapping emission spectra arefound, the set may still not be suitable if the respective fluorescentefficiencies are too low. For example, in the case of DNA sequencing,increased sample loading can not compensate for low fluorescenceefficiencies, Pringle et al., DNA Core Facilities Newsletter, 1:15-21(1988). Third, when several fluorescent dyes are used concurrently,simultaneous excitation becomes difficult because the absorption bandsof the dyes are widely separated. Fourth, the charge, molecular size,and conformation of the dyes must not adversely affect theelectrophoretic mobilities of the fragments. Finally, the fluorescentdyes must be compatible with the chemistry used to create or manipulatethe fragments, e.g., DNA synthesis solvents and reagents, buffers,polymerase enzymes, ligase enzymes, and the like.

Because of these severe constraints only a few sets of fluorescent dyeshave been found that can be used in multicolor applications,particularly in the area of four-color DNA sequencing, e.g., Smith etal., Nucleic Acids Research, 113; 2399-2412 (1985); Prober et al.,Science, 238: 336341 (1987); and Connell et al., Biotechniques, 5:342-348 (1987). FIG. 1 shows examples of fluorescent xanthene dyescurrently used as long-wavelength labels emitting above 550 nm includingthe two rhodamine-based dyes TAMRA (22) and ROX (26) and the twofluorescein-based dyes HEX (23) and NAN (24).

SUMMARY

The present invention is directed towards our discovery of a class ofasymmetric benzoxanthene dyes useful as fluorescent dyes.

It is an object of our invention to provide a class of asymmetricbenzoxanthene dyes useful for the simultaneous detection of multiplespatially-overlapping analytes which satisfies the constraints describedabove and provide fluorescence emission maxima above 550 nm whenilluminated by excitation light having a wavelength of between 480 nmand 550 nm

It is a further object of our invention to provide a class of asymmetricbenzoxanthene dyes useful for the simultaneous detection of multiplespatially overlapping analytes which satisfies the constraints describedabove and whose fluorescence properties may be tuned by manipulation ofsubstituents at a variety of positions.

It is another object of our invention to provide methods andintermediate compounds useful for the synthesis of the asymmetricbenzoxanthene dyes of our invention.

It is a further object of our invention to provide nucleotides andpolynucleotides labeled with the asymmetric benzoxanthene dyes of ourinvention.

It is another object of our invention to provide phosphoramiditecompounds including the asymmetric benzoxanthene dyes of our invention.

It is another object of our invention to provide fragment analysismethods, including DNA sequencing methods, employing the asymmetricbenzoxanthene dyes of our invention.

In a first aspect, the foregoing and other objects of our invention areachieved by an asymmetric benzoxanthene dye compound having the formula.##STR3## wherein Y₁ and Y₂ taken separately are hydroxyl, oxygen,imminium, or amine. R₁ -R₈ taken separately are hydrogen, fluorine,chlorine, lower alkyl, lower alkene, lower alkyne, sulfonate, amino,ammonium, amido, nitrile, alkoxy, linking group, or combinationsthereof. And, R₉ is acetylene, alkane, alkene, cyano, substitutedphenyl, or combinations thereof, the substituted phenyl having thestructure: ##STR4## wherein X₁ is carboxylic acid or sulfonic acid; X₂and X₅ taken separately are hydrogen, chlorine, fluorine, or lowerallyl; and X₃ and X₄ taken separately are hydrogen, chlorine, fluorine,lower alkyl, carboxylic acid, sulfonic acid, or linking group.

In a second aspect, the invention includes phosphoramidite compoundshaving the formula: ##STR5## wherein X is a spacer arm; Y is a linkage;B₁ is a phosphite ester protecting group; B₂, and B₃ taken separatelyare selected from the group consisting of lower alkyl, lower alkene,lower aryl having between 1 and 8 carbon atoms, arylalkyl, andcycloalkyl containing up to 10 carbon atoms; and D is the asymmetricbenzoxanthene dye compound described above. Y and D are linked through alinkage formed by the reaction of a linking group and its complementaryfunctionality, such linkage being attached to dye D at one of positionsR₁ -R₉.

In a third aspect, the invention includes a phosphoramidite compoundhaving the formula: ##STR6## wherein B₁ is a phosphite ester protectinggroup, B₂ and B₃ taken separately are selected from the group consistingof lower allyl lower alkene, lower aryl having between 1 and 8 carbonatoms, arylalkyl and cycloalkyl containing up to 10 carbon atoms; B₅ isan acid-cleavable hydroxyl protecting group; B is a nucleotide base; andD is the dye compound described above. When B is purine or7-deazapurine, the sugar moiety is attached at the N⁹ -position of thepurine or 7-deazapurine, and when B is pyrimidine, the sugar moiety isattached at the N¹ -position of the pyrimidine. B and D are linkedthrough a linkage formed by the reaction of a linking group and itscomplementary functionality, such linkage being attached to D at one ofpositions R₁ -R₉. If B is a purine, the linkage is attached to the8-position of the purine, if B is 7-deazapurine, the linkage is attachedto the 7-position of the 7-deazapurine, and if B is pyrimidine, thelinkage is attached to the 5-position of the pyrimidine. Preferably B isselected from the group consisting of uracil, cytosine, 7-deazapurine,and 7-deazaguanosine.

In a fourth aspect, the present invention includes a compound useful asan intermediate in the synthesis of the above described asymmetricbenzoxanthene dyes, such compound having the formula: ##STR7## whereinR₃ -R₇ are as described above and Y₂ is hydroxyl or amine. In aparticularly preferred embodiment of this aspect, R₃ is fluorine and Y₂is hydroxyl.

In a fifth aspect the invention includes a nucleotide labeled with theabove described asymmetric benzoxanthene dyes of the invention, thenucleotide having the formula: ##STR8## wherein B is a 7-deazapurine,purine, or pyrimidine nucleotide base; W₁ and W₂ taken separately are Hor OH; W₃ is OH, ##STR9## and, D is a dye compound of the invention.When B is purine or 7-deazapurine, the sugar moiety is attached at theN⁹ -position of the purine or deazapurine, and when B is pyrimidine, thesugar moiety is attached at the N¹ -position of the pyrimidine. Thelinkage linking B and D is attached to D at one of positions R₁ -R₉. IfB is a purine, the linkage is attached to the 8-position of the purine,if B is 7-deazapurine, the linkage is attached to the 7-position of the7-deazapurine, and if B is pyrimidine, the linkage is attached to the5-position of the pyrimidine. Preferably B is selected from the groupconsisting of uracil cytosine, deazaadenine, and deazaguanosine.

In a sixth aspect, the invention includes labeled polynucleotidescontaining a nucleotide having the formula: ##STR10## wherein B is a7-deazapurine, purine, or pyrimidine nucleotide base; Z, is H or OH; Z₂is H; OH; HPO₄, and Nuc, wherein "Nuc" refers to a nucleotide. Thenucleoside and Nuc are linked by a phosphodiester linkage, the linkagebeing attached to the 5'-position of Nuc; Z₃ is selected from the groupconsisting of H, HPO₃ and phosphate analogs thereof, and Nuc, whereinNuc and the nucleoside are linked by a phosphodiester linkage, thelinkage being attached to the 3'-position of Nuc; and D is a dyecompound of the invention Phosphate analogs of HPO₃ include analogswherein a non-bridging oxygen is replaced by a non-oxygen moiety, e.g.,sulphur, amino, anilidate, anilinthioate, and the like.

When B is purine or 7-deazapurine, the sugar moiety is attached at theN⁹ -position of the purine or deazapurine, and when B is pyrimidine, thesugar moiety is attached at the N¹ -position of the pyrimidine. Thelinkage linking B and D is attached to D at one of positions R₁ -R₉. IfB is a purine, the linkage is attached to the 8-position of the purine,if B is 7-deazapurine, the linkage is attached to the 7-position of the7-deazapurine, and if B is pyrimidine, the linkage is attached to the5-position of the pyrimidine. Preferably B is selected from the groupconsisting of uracil, cytosine, deazaadenine, and deazaguanosine.

In a seventh aspect, the invention includes a method of polynucleotidesequencing using the dyes of the invention. The method comprises thesteps of forming a mixture of a first, a second, a third, and a forthclass of polynucleotides such that each polynucleotide in the firstclass includes a 3'-terminal dideoxyadenosine and is labeled with afirst dye; each polynucleotide in the second class includes a3'-terminal dideoxycytidine and is labeled with a second dye; eachpolynucleotide in the third class includes a 3'-terminaldideoxyguanosine and is labeled with a third dye; and eachpolynucleotide in the forth class includes a 3'-terminal dideoxymidineand is labeled with a forth dye. In the method, one or more of the firstsecond, third, or forth dyes is an asymmetric benzoxanthene dye of theinvention. The other of the dyes is chosen such that they are spectrallyresolvable from the asymmetric benzoxanthene dye(s) and from each other.After forming the above mixture, the polynucleotides areelectrophoretically separated thereby forming bands of similarly sizedpolynucleotides. Next, the bands are illuminated with an illuminationbeam capable of causing the dyes to fluoresce. Finally, the classes ofthe polynucleotides are identified by the fluorescence spectrum of thelabeled polynucleotides in each band.

In an eighth aspect, the invention includes a method of fragmentanalysis utilizing the dye compounds of the present invention. Themethod of this aspect comprises the steps of forming a labeledpolynucleotide fragment, the fragment being labeled with a dye compoundof the invention; subjecting the labeled polynucleotide fragment to asize-dependent separation process; and detecting the labeledpolynucleotide fragment subsequent to the separation process.

The dyes of the present invention provide at least seven importantadvantages over currently available dyes used for the simultaneousdetection of multiple spatially-overlapping analytes, particularly inthe area of multicolor fluorescence-based DNA sequencing. First, thedyes of the present invention are much more stable to DNA synthesisconditions then are presently available dyes having the desired spectralcharacteristics. This enhanced stability to DNA synthesis conditionsmakes it possible to more readily prepare labeled oligonucleotidereagents using automated DNA synthesis technologies, e.g., labeled PCRprimers, DNA sequencing primers, and oligonucleotide hybridizationprobes. Second, the dyes of the present invention are significantly morephotostable than fluorescein-based dyes previously employed in thewavelength region above about 550 nm. Third, the dyes of the presentinvention have an absorption spectrum which has a blue "shoulder"thereby permitting more efficient excitation of the dyes at shorterwavelengths than dibenzoxanthene dyes or rhodamine-based dyes. Fourth,the asymmetric benzoxanthene dyes of the present invention havesignificantly higher quantum yields then do spectrally similarrhodamine-based dyes. Fifth, the enhanced excitation efficiency withtypical light sources coupled with the high quantum yields of the dyesof the present invention make the dyes significantly brighter thanpresently available dyes having the desired spectral characteristics.Brightness is particularly important in the context of DNA sequencingapplications where the amount of analyte is limited by electrophoresisloading factors and the total fluorescence is distributed over hundredsof spatially separated species. As used herein the term "brightness"refers to the combined effects of extinction coefficient andfluorescence quantum yield on ultimate fluorescence emission intensity.By increasing the brightness of the fluorescent labels, the larger, lessabundant fragments can be more readily detected and less sample need beloaded into the electrophoresis, thereby resulting in superiorelectrophoretic resolution. Moreover, the increased brightness of theanalytes contributes to increased signal-to-noise ratio leading toimproved deconvolution of spatially and spectrally neighboring species.Sixth, the asymmetry of the dyes of the present invention permits tuningof the emission spectrum of the dyes by varying the substituents R₁ -R₉,particularly substituents R₁ -R₃ on the resorcinol-derived portion ofthe dye. Only one equivalent substituent position is available onsymmetric dibenzoxanthene compounds, thereby greatly limiting thedegrees of freedom available for spectral tuning of the dyes. Seventh,the dyes of the invention are readily converted to stablephosphoramidite derivatives which can be employed in the automatedchemical synthesis of labeled oligonucleotides.

These and other objects, features, and advantages of the presentinvention will become better understood with reference to the followingdescription, drawings, and appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the structures of various fluorescent dyes previouslyemployed as long-wavelength labels, i.e., labels emitting above 550 nm.

FIGS. 2A and 2B depict a preferred synthesis of the asymmetricbenzoxanthene dyes of the invention.

FIG. 3 shows a preferred synthesis of oligonucleotides labeled with thedyes of the invention.

FIG. 4 shows the excitation spectra of TAMRA (22) - and Cl-FLAN(2)-labeled oligonucleotides.

FIG. 5 shows a comparison of the quantum yields of TAMRA (22) - andCl-FLAN (2)-labeled oligonucleotides.

FIG. 6 shows a comparison of the eqimolar emission intensity of TAMRA(22) - and Cl-FLAN (2)-labeled oligonucleotides.

FIG. 7 shows fluorescence emission spectra for members of a 4-plex setof dye-labeled DNA sequencing primers.

FIG. 8 shows a synthesis of a 2-fluoro-1,3-dihydroxynapthaleneintermediate of the invention.

FIG. 9 shows the results of a DNA sequencing experiment employing anoligonucleotide sequencing primer labeled with a dye compound of theinvention.

FIG. 10 shows the results of a microsatellite analysis employing anoligonucleotide PCR primer labeled with a dye compound of the invention.

FIG. 11 shows four preferred synthesis routes for the synthesis of theasymmetric benzoxanthene dyes of the invention.

FIG. 12 shows three preferred synthesis routes for the synthesis of the1-substituted, 3-hydroxynapthalene intermediate of the invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Reference will now be made in detail to certain preferred embodiments ofthe invention. While the invention will be described in conjunction withthe preferred embodiments, it will be understood that they are notintended to limit the invention to those embodiments. On the contrary,the invention is intended to cover alternatives, modifications, andequivalents, which may be included within the invention as defined bythe appended claims.

Generally, the present invention comprises a novel class of asymmetricbenzoxanthene compounds useful as fluorescent dyes, methods andintermediates for synthesis of such dyes, reagents employing such dyesas molecular labels, and methods utilizing such dyes and reagents in thearea of analytical biotechnology. The compounds of the present inventionfind particular application in the area of multicolor fluorescent DNAsequencing and fragment analysis.

I. Asymmetric Benzoxanthene Dye Compounds

In a first aspect, the present invention comprises a novel class ofasymmetric benzoxanthene dye compounds having the general structureshown in Formula I immediately below. (All molecular structures providedherein are intended to encompass not only the exact electronic structurepresented, but also include all resonant structures and protonationstates thereof.) ##STR11##

In Formula I, Y₁ and Y₂ are either individually hydroxyl, oxygen, amine,imminium or oxygen. When Y₁ is hydroxyl and Y₂ is oxygen, the compoundis analogous to fluorescein, while when Y₁ is amine and Y₂ is imminium,the compound is analogous to rhodamine. Preferably Y₁ is hydroxyl and Y₂is oxygen.

Moieties R₁ -R₉ are substituents used to modulate various properties ofthe dyes by modifying the electronic structure of the ground and excitedstates of the molecule. In particular, varying moieties R₁ -R₉ affectsthe spectral characteristics, chemical stability, and photostability ofthe compounds. Substituents R₁ -R₃ and R₉ are particularly important indefining the properties of the compounds of Formula I. For example, ithas been observed that placing a fluorine atom at one of positions R₁-R₃ leads to increased chemical and photostability, and that if R₉ issubstituted phenyl, making substituents X₂ and X₅ chlorine leads tonarrower emission bands. (See below for the definition of substituentsX₂ and X₅.)

Preferably, substituents R₁ -R₈ are hydrogen, fluorine, chlorine, loweralkyl, lower alkene, lower alkyne, sulfonate, sulfone, amino, imminium,amido, nitrile, aryl lower alkoxy, linking group, or combinationsthereof where as used herein the term "linking group" refers to afunctionality capable of reacting with a "complementary functionality"attached to a reagent, such reaction forming a "linkage" connecting thedye to the reagent. More will be said about particular linking groups,complementary functionalities, and linkages in a subsequent section ofthis disclosure. Preferably, R₁ is lower alkoxy, chlorine, fluorine, orhydrogen; R₂ is lower alkyl, fluorine, or chlorine; and R₃ is loweralkyl or fluorine. More preferably, one of R₁, R₂, and R₃ is fluorine.In a particularly preferred embodiment, at least R₃ is fluorine.

As used herein, the term "lower alkyl" denotes straight-chain andbranched hydrocarbon moieties containing from 1 to 8 carbon atoms, i.e.,methyl ethyl, propyl isopropyl tert-butyl isobutyl, sec-butyl neopentyltert-pentyl, and the like. "Lower substituted alkyl" denotes a loweralkyl including electron-withdrawing substituents, such as halo, cyano,nitro, sulfo, or the like. "Lower haloalkyl" denotes a lower substitutedalkyl with one or more halogen atom substituents, usually fluoro,chloro, bromo, or iodo. "Lower alkene" denotes a hydrocarbon containingfrom 1 to 8 carbon atoms wherein one or more of the carbon--carbon bondsare double bonds, wherein the non-double bonded carbons comprise loweralkyl or lower substituted alkyl. "Lower alkyne" denotes a hydrocarboncontaining from 1 to 8 carbon atoms wherein one or more of the carbonsare bonded with a triple bond, wherein the non-triple bonded carbonscomprise lower alkyl or lower substituted alkyl. "Sulfonate" refers tomoieties including a sulfur atom bonded to 3 oxygen atoms, includingmono- and di-salts thereof e.g., sodium sulfonate, potassium sulfonate,disodium sulfonate, and the like. "Amino" refers to moieties including anitrogen atom bonded to 2 hydrogen atoms, lower alkyl moieties, or anycombination thereof. "Amido" refers to moieties including a carbon atomdouble bonded to an oxygen atom and single bonded to an amino moiety."Nitrile" refers to moieties including a carbon atom triple bonded to anitrogen atom. "Lower Alkoxy" refers to a moiety including lower alkylsingle bonded to an oxygen atom. "Aryl" refers to single or multiplephenyl or substituted phenyl e.g., benzene, naphthalene, anthracene,biphenyl and the like.

Preferably R₉ is acetylene, lower alkyl lower alkene, cyano, phenyl orsubstituted phenyl, heterocyclic aromatic, or combinations thereof thesubstituted phenyl having the structure: ##STR12## wherein X₁ -X₅ takenseparately are hydrogen, chlorine, fluorine, lower alkyl carboxylicacid, sulfonic acid, --CH₂ OH, or linking group. As used herein, theterm "heterocyclic aromatic" refers to aromatic moieties having aheteroatom as part of the cyclic structure, e.g., pyrole, furan, indole,and the like. Preferably, X₁ is carboxylic acid, sulfonic acid, or --CH₂OH; X₂ and X₅ taken separately are hydrogen, chlorine, fluorine, orlower alkyl and X₃ and X₄ taken separately are hydrogen, chlorine,fluorine, lower allyl carboxylic acid, sulfonic acid, or lining group.More preferably, X₂ and X₅ are chlorine. In an additional preferredembodiment, one of X₃ or X₄ is linking group. Preferably, X₁ iscarboxylic acid. In an additional preferred embodiment particularlysuited to forming phosphoramidite compounds including the dye compoundof the invention, one of X₁ or X₅ is a moiety which is capable offorming a cyclic ester or cyclic ether, e.g., carboxylic acid, sulfonicacid, or --CH₂ OH, or any other group that will form a spirocyclicsystem, i.e., bicyclic compounds having one carbon atom common to bothrings, e.g., spiro[4.5]decane.

Preferably the linking group of the invention is isothiocyanate,sulfonyl chloride, 4,6-dichlorotriazinylanine, succinimidyl ester, orother active carboxylate whenever the complementary functionality isamine. Preferably the linking group is maleimide, halo acetyl, oriodoacetamide whenever the complementary functionality is sulfhydryl.See R. Haugland, Molecular Probes Handbook of Fluorescent Probes andResearch Chemicals, Molecular probes, Inc. (1992). In a particularlypreferred embodiment, the lining group is an activatedN-hydroxysuccinimidyl (NHS) ester which reacts with an aminecomplementary functionality, where to form the activated NHS ester, adye of the invention including a carboxylate linking group is reactedwith dicyclohexylcarbodiimide and N-hydroxysuccinimide to form the NHSester. See FIG. 3.

Several alternative generalized methods may be used to synthesize theasymmetric benzoxanthene dye compounds of the present invention, four ofwhich will be described here with reference to FIG. 11. In a firstpreferred method referred to in FIG. 11 as Route A, compound 27 isreacted with 1,3-dihydroxy or 1,3-aminohydroxy benzene derivative 28 and1,3-dihydroxy or 1,3-aminohydroxy naphthalene derivative 29 employingequal equivalents of each under acid catalysis and heat to giveasymmetric dye compound 30. Preferably compound 27 is a cyclic orstraight chain anhydride, e.g., LVG is OCOR₉ ; ester, e.g., where LVG isOR where R is lower alkyl, phenyl, or sulfonate; or acid chloride, e.g.,where LVG is chlorine or other halogen.

In an alternative preferred synthesis method referred to as Route B inFIG. 11, compound 27 is reacted with 2 equivalents of a1,3-dihydroxybenzene derivative, i.e., Y₁ is hydroxy, or a1,3-aminohydroxybenzene derivative, i.e., Y₁ is amino, 28 to givesymmetric xanthene dye 31. Compound 31 is then decomposed by basehydrolysis to form intermediate benzoyl condensation product 32.Condensation product 32 is then reacted under acid catalysis and heatwith compound 29 to give asymmetric dye 30, where 29 is1,3-dihydroxynapthalene when Y₂ is hydroxy, or1,3-aminohydroxynaphthalene when Y₂ is amino.

In yet a third generalized synthesis method, referred to as Route C inFIG. 11, compound 27 is reacted with 1 equivalent of 28 with heat togive intermediate benzoyl condensation product 32. Compound 32 is thenreacted with 29 under acid catalysis and heat to give asymmetric dye 30.

In a fourth generalized synthesis method, referred to as Route D in FIG.11, equal equivalents of compound 33, compound 28, and compound 29 arereacted under acid catalysis and heat to give asymmetric xanthoneintermediate 34. Preferably 33 is a carbonate, e.g., LVG is OR where Ris preferably lower alkyl or phenyl; or formate, e.g., where LVG ishalogen and OR where R is preferably lower alkyl or phenyl. Compound 34is then reacted with an anionic organometallic R₉ derivative to give theasymmetric dye 30, e.g., R₉ Li R₉ MgX where X is halide, e.g., Br, Cl,I, and the like.

FIGS. 2A and 2B show the synthesis of a set of particularly preferredasymmetric dye compounds of the invention. In this synthesis, a 1,3dihydroxynapthalene derivative, such as 1,3 dihydroxynapthalene (9b) or2-fluoro-1,3-dihydroxynapthalene (9a), is reacted with 1 equivalent of aphthallic anhydride derivative, e.g., 3,6-dichlorotrimelletic acidanhydride (10a), and one equivalent of a resorcinol derivative (11a,11b, 11e, or 11d), and heated for 16 hours in neat organic acid, e.g.,MeSO₃ H under argon. The crude dye is then precipitated by addition toan ice / water mixture and isolated by filtration. The crude dye is thenfurther purified into 2 isomers 1 and 2 by preparative thin layerchromatography.

Unsubstituted derivatives of the asymmetric benzoxanthene dyes (R₂and/or R₃ is H) may be reacted further with halogenating reagents, e.g.,commercially available sources of positive fluorine, NaOCl, NaOH / Br₂,NaOH / I₂, to produce quantitatively halogenated derivatives, e.g., R₂=R₃ =Cl, Br, L or F after extractive workup with 10% HCl / EtOAc, dryingwith Na₂ SO₄, filtering, and concentrating in vacuo. See inset in FIG.2B.

II. Substituted Naphthalene Intermediates

In a second aspect, the present invention comprises novel intermediatecompounds useful for the synthesis of the asymmetric benzoxanthenecompounds of the subject invention, such intermediate having the generalstructure shown in Formula II immediately below. In particular, theintermediate compounds of the invention enable the synthesis ofasymmetric benzoxanthene compounds with regio-selective incorporation ofsubstituents, e.g., halogen atoms, at the 2-position of 2-substitutedasymmetric benzoxanthene compounds, where the 2-position corresponds tothe R₃ position in the compounds of Formulas I and II. ##STR13##Substituents R₃ -R₇ in the structure of Formula II correspond tolike-numbered substituents in the structure of Formula I describedabove, and Y₂ is hydroxyl or amine. Preferably, R₃ is fluorine and Y₂ ishydroxyl.

FIG. 12 shows three alternative generalized synthesis schemes for thesynthesis of the substituted naphthalene intermediates of the invention.In a first method indicated as Route A in FIG. 12, substituted esterenolate derivative 35 is reacted with activated homophthamic acid esterderivative 36 to give β-keto-ester derivative 37, e.g., by spontaneousloss of CO₂ when R' is carboxylate. Preferably in compound 35, R' ishydrogen, carboxylate, or halogen and R is lower alkyl. Preferably incompound 36, LVG is halogen, N-hydroxysuccinimide, phenoxide,hydroxybenzotriazole, or carboxylate. Compound 37 is then cyclized underbase catalysis and heat to give substituted 1,3-naphthalene diol 38,i.e., Y₂ is OH.

In a second preferred synthesis method indicated as Route B in FIG. 12,compound 35 is reacted with activated phenylacetate derivative 39, whereLVG is as described above for compound 36 in Route A, to giveβ-keto-ester derivative 40, e.g., by spontaneous loss of CO₂ when R' iscarboxylate. Compound 40 is then cyclized under acid catalysis and heatto give substituted 1,3-naphthalene diols 38, i.e., Y₂ is OH.

In a third preferred synthesis method indicated as Route C, compound 35is reacted with cyano-phenyl acetate derivatives 41, where LVG is asdescribed above for compound 36 in Route A, to give cyano β-keto-esterderivatives 42, e.g., by spontaneous loss of CO₂ when R' is carboxylate.Compound 42 is then cyclized under base catalysis and heat to givesubstituted 1-amino-3-hydroxynaphthalenes 38, i.e., Y₂ is NH₂.

III. Regeants Utilizing Dye Compounds

In another aspect, the present invention comprises reagents labeled withthe asymmetric benzoxanthene dye compounds of Formula I. Reagents of theinvention can be virtually anything to which the dyes of the inventioncan be attached. Preferably the dyes are covalently attached to thereagent. Reagents include proteins, polypeptides, polysaccharides,nucleotides, nucleosides, polynucleotides, lipids, solid supports,organic and inorganic polymers, and combinations and assemblages thereofsuch as chromosomes, nuclei living cells, such as bacteria, othermicroorganisms, mammalian cells, tissues, and the like.

A. Nucleotide Reagents

A preferred class of reagents of the present invention comprisenucleotides and nucleosides which incorporate the asymmetricbenzoxanthene dyes of the invention Such nucleotide reagents areparticularly useful in the context of labeling polynucleotides formed byenzymatic synthesis, e.g., nucleotide triphosphates used in the contextof PCR amplification, Sanger-type polynucleotide sequencing, andnick-translation reactions.

As used herein, "nucleoside" refers to a compound consisting of apurine, deazapurine, or pyrimidine nucleoside base, e.g., adenine,guanine, cytosine, uracil, thymine, deazapurine, deazaguanosine, and thelike, linked to a pentose at the 1' position, including 2'-deoxy and2'-hydroxyl forms, e.g. as described in Komberg and Baker, DNAReplication, 2nd Ed. (Freeman, San Francisco, 1992). The term"nucleotide" as used herein refers to a phosphate ester of a nucleoside,e.g., triphosphate esters, wherein the most common site ofesterification is the hydroxyl group attached to the C-5 position of thepentose. "Analogs" in reference to nucleosides include syntheticnucleosides having modified base moieties and/or modified sugarmoieties, e.g. described generally by Scheit, Nucleotide Analogs (JohnWiley, New York, 1980). The term "labeled nucleoside" refers tonucleosides which are covalently attached to the dye compounds ofFormula I through a linkage.

Preferred nucleotides of the present invention are shown below inFormula III wherein ##STR14## B is a nucleotide base, e.g., uracilcytosine, deazaadenine, and deazaguanosine. W₁ and W₂ taken separatelyare H or OH W₃ is OH, ##STR15## including associated counterions ifpresent, e.g., H, Na, NH₄, and the like. D is a dye compound of FormulaI. In one particularly preferred embodiment, the nucleotides of thepresent invention are dideoxynucleotide triphosphates having thestructure shown in Formula III.1 below, including associated counterionsif present. ##STR16## Labeled dideoxy nucleotides such as that shown inFormula III.1 find particular application as chain terminating agents inSanger-type DNA sequencing methods. In a second particularly preferredembodiment, the nucleotides of the present invention are deoxynucleotidetriphosphates having the structure shown in Formula III.2 below,including associated counterions if present. ##STR17## Labeleddeoxynucleotides such as that shown in Formula III1.2 find particularapplication as means for labeling polymerase extension products, e.g.,in the polymerase chain reaction.

When B is purine or 7-deazapurine, the sugar moiety is attached at theN⁹ -position of the purine or deazapurine, and when B is pyrimidine, thesugar moiety is attached at the N¹ -position of the pyrimidine.

The linkage linking B and D is attached to D at one of positions R₁ -R₉. Preferably, the linkage is not attached at R₁ -R₃. When the dyes ofthe invention are synthesized from trimelletic anhydride, R₉ ispreferably substituted phenyl and the linkage is attached to the dye atone of the X₃ or X₄ positions of the substituted phenyl, the otherposition being a hydrogen atom.

When B is a purine, the linkage linking B and D is attached to the8-position of the purine, when B is 7-deazapurine, the linkage isattached to the 7-position of the 7-deazapurine, and when B ispyrimidine, the linkage is attached to the 5-position of the pyrimidine.

Nucleoside labeling can be accomplished using any of a large number ofknown nucleoside labeling techniques using known linkages, lininggroups, and associated complementary functionalities. The linkagelinking the dye and nucleoside should (i) be stable to oligonucleotidesynthesis conditions, (ii) not interfere with oligonucleotide-targethybridization, (iii) be compatible with relevant enzymes, e.g.,polymerases, ligases, and the like, and (iv) not quench the fluorescenceof the dye.

Preferably, the dyes are covalently linked to the 5-carbon of pyrimidinebases and to the 7-carbon of 7-deazapurine bases. Several suitable baselabeling procedures have been reported that can be used with theinvention, e.g. Gibson et al, Nucleic Acids Research, 15:6455-6467(1987); Gebeyehu et al, Nucleic Acids Research, 15: 4513-4535 (1987);Haralambidis et al, Nucleic Acids Research, 15: 4856-4876 (1987); Nelsonet al., Nucleosides and Nucleotides, 5(3): 233-241 (1986); Bergstrom, etal., JACS, 111: 374-375 (1989); U.S. Pat. Nos. 4,855,225, 5,231,191, and5,449,767. Accordingly, these references are incorporated by reference.

Preferably, the linkages are acetylenic amido or alkenic amido linkages,the linkage between the dye and the nucleotide base being formed byreacting an activated N-hydroxysuccinimide (NHS) ester of the dye withan alkynylamino- or alkenylamino-derivatized base of a nucleotide. Morepreferably, the resulting linkage is 3-(carboxy)amino-1-propynyl or3-amino-1-propyn-1-yl (Formula III.3). Several preferred linkages forlinking the dyes of the invention to a nucleoside base are shown belowin Formulas III.3, III.4, and III.5. ##STR18##

The synthesis of alkynylamino-derivatized nucleosides is taught by Hobbset al. in European Patent Application No. 87305844.0, and Hobbs et al.,J. Org. Chem., 54: 3420 (1989), which is incorporated herein byreference. Briefly, the alkynylamino-derivatized nucleotides are formedby placing the appropriate halodideoxynucleoside (usually5-iodopyrimidine and 7-iodo-7-deazapurine dideoxynucleosides as taughtby Hobbs et al. (cited above)) and Cu(I) in a flask, flushing with argonto remove air, adding dry DMF, followed by addition of an alkynylamine,triethyl-amine and Pd(0). The reaction mixture can be stirred forseveral hours, or until thin layer chromatography indicates consumptionof the halodideoxynucleoside. When an unprotected alkynylamine is used,the allynylamino-nucleoside can be isolated by concentrating thereaction mixture and chromatographing on silica gel using an elutingsolvent which contains ammonium hydroxide to neutralize the hydrohalidegenerated in the coupling reaction When a protected allynylamine isused, methanol/methylene chloride can be added to the reaction mixture,followed by the bicarbonate form of a strongly basic anion exchangeresin. The slurry can then be stirred for about 45 minutes, filtered,and the resin rinsed with additional methanol/methylene chloride. Thecombined filtrates can be concentrated and purified byflash-chromatography on silica gel using a methanol-methylene chloridegradient. The triphosphates are obtained by standard techniques.

B. Phosphoramidite Reagents

Another preferred class of reagents comprise phosphoramidite compoundswhich incorporate the asymmetric benzoxanthene dyes of the invention.Such phosphoramidite reagents are particularly useful for the automatedchemical synthesis of polynucleotides labeled with the asymmetricbenzoxanthene dyes of the invention. Such phosphoramidite compounds whenreacted with a 5'-hydroxyl group of a nucleotide or polynucleotide forma phosphite ester linker which, in turn, is oxidized to give a phosphateester linker, e.g., U.S. Pat. Nos. 4,458,066 and 4,415,732, both patentshereby incorporated by reference.

1. Non-nucleotide Phosphoramidite Reagents: Generally, in one aspect,the phosphoramidite reagents of the invention have the structure ofFormula IV immediately below, ##STR19## where X is a spacer arm; D is anasymmetric benzoxanthene dye of Formula I or a protected derivativethereof, Y is a linkage formed with a linking group on the dye; B, is aphosphite ester protecting group, and B₂, and B₃ taken separately arelower alkyl, lower alkene, lower aryl having between 1 and 8 carbonatoms, aralkyl, or cycloalkyl containing up to 10 carbon atoms.Non-nucleotidic phosphoramidites as shown in Formula IV are particularlywell suited for labeling the 5'-end of a chemically-synthesizedpolynucleotide through the sugar-portion of the nucleotide.

Spacer X and linkage Y may take a variety of forms, however, thestructure X-Y must be such that (i) it is stable to DNA synthesisconditions, (ii) does not interfere with oligonucleotide-targethybridization, and (iii) does not quench the fluorescence of the dye towhich it is attached, e.g., U.S. Pat. Nos. 5,231,191, 5,258,538, and4,757,141, 5,212,304, all patents hereby incorporated by reference.

Preferably X is linear or cyclic lower alkyl linear or cyclicsubstituted lower alkyl, polyethlene oxide, lower aryl having between 1and 8 carbon atoms, peptide, or polyether. Preferably the linkage Y isamido, sulfonamido, urea, urethane, or thourea. In one particularlypreferred embodiment, the linkage Y is amido and the spacer X is linearalkyl having the structure below in Formula IV.1 ##STR20## where n isfrom 2 to 30, preferably from 2 to 10, and more preferably from 2 to 6.In a second particularly preferred embodiment, the linkage Y is amidoand the spacer X is linear polyethylene oxide having the structure shownbelow in Formula IV.2 ##STR21## where n is from 2 to 30, preferably from2 to 10, and more preferably from 2 to 6.

Preferably, B₂ and B₃ taken together form an alkyl chain containing upto 5 carbon atoms in the principle chain and a total of up to 10 carbonatoms with both terminal valence bonds of said chains being attached tothe nitrogen atom. Alternatively, B₂ and B₃ taken together with thenitrogen atom form a saturated nitrogen heterocycle which contains oneor more heteroatoms selected from the group consisting of nitrogen,oxygen, and sulfur. Preferably, B₂ and B₃ taken separately areisopropyl, t-butyl, isobutyl or sec-butyl, and B₂ and B₃ taken togetheris morphollino.

B₁ is a phosphite ester protecting group which prevents unwantedextension of the polynucleotide to which the phosphoramidite isattached. B₁ is stable to polynucleotide synthesis conditions yet isable to be removed from the polynucleotide product with a reagent thatdoes not adversely affect the integrity of the polynucleotide or thedye. Preferably, B₁ is methyl, β-cyanoethyl, or 4-nitrophenylethyl. B₂and B₃ taken separately are isopropyl, t-butyl, isobutyl or sec-butyl,and B₂ and B₃ taken together is morphollino.

The linkage linking Y and D is attached to D at one of positions R₁ -R₉.Preferably, the linkage is not attached at R₁ -R₃. When the dyes of theinvention are synthesized from trimelletic anhydride, R₉ is preferablysubstituted phenyl and the linkage is attached to the dye at one of theX₃ or X₄ positions of the substituted phenyl.

Such phosphoramidite compounds may be synthesized by known methods.Generally, the synthesis proceeds as follows. Phenolic hydroxyls of thedye are protected with dye-protecting groups that can be removed with aDNA synthesis deprotection agent, e.g., ammonia, ethanolamine,methylamine/ammonium hydroxide mixtures, and mixtures oft-butylamine/water/methanol (1:2:1), e.g., see U.S. Pat. No. 5,231,191,hereby incorporated by reference in its entirety. Dyes so protected arereferred to herein as "protected derivatives" of the dye. Preferredprotecting groups include esters of benzoic acid or pivalic acid. Thelinking group of the protected dye, e.g., carboxylic acid, is thenactivated, e.g., with carbodiimide, and reacted with an alcohol linkerderivative, e.g., an amino alcohol e.g., ethanolamine, hexanol amine, orthe like, in N,N diethylformamide (DMF), or another like aprotic solventto yield a protected dye with a free alcohol functionality, e.g.,alcohol-amide derivative. The free alcohol is then reacted with aphosphitylating agent using standard procedures, e.g.,di-(N,N-diisopropylamino)methoxyphosphine in acetonitrile containingcatalytic amounts of tetrazole diisopropylamine, to yield thephosphoramidite, e.g., U.S. Pat. No. 5,231,191.

2. Nucleotidic Phosphoramidite Reagents: Generally, in a second aspect,the phosphoramidite reagents of the invention have the structure ofFormula V immediately below, ##STR22## where B₁ -B₃ are as describedabove, B₅ is hydrogen or a hydroxyl protecting group, B is a nucleotidebase, and D is an asymmetric benzoxanthene dye of Formula I, or aprotected derivative thereof. Nucleotide phosphoramidites such as shownin Formula V are particularly well suited for the internal labeling ofchemically-synthesized polynucleotides.

When B is purine or 7-deazapurine, the sugar moiety is attached at theN⁹ -position of the purine or deazapurine. Alternatively, when B ispyrimidine, the sugar moiety is attached at the N¹ -position of thepyrimidine. B and D are linked through a linkage formed by the reactionof a linking group and its complementary functionality, such linkagesbetween dyes and nucleotide bases have been described in detail above.If B is a purine, the linkage is attached to the 8-position of thepurine, while if B is 7-deazapurine, the linkage is attached to the7-position of the 7-deazapurine. If B is pyrimidine, the linkage isattached to the 5-position of the pyrimidine.

B₅ refers generally to hydrogen or an acid-cleavable hydroxyl protectinggroup. Preferably, B₅ is the triphenylmethyl radical and itselectron-donating-substituted derivatives, where, as used herein, theterm "electron-donating" denotes the tendency of a substituent torelease valence electrons to neighboring atoms in the molecule of whichit is a part, i.e., it is electropositive with respect to neighboringatoms. Preferably, electron-donating substituents include amino, loweralkyl, lower aryl having between 1 and 8 carbon atoms, lower alkoxy, andthe like. More preferably, the electron-donating substituents aremethoxy. Exemplary trityls include 4,4'-dimethoxytrityl, i.e.bis(p-anisyl)phenylmethyl, monomethoxytrityl, α-naphthyldiphenylmethyl,tri(p-methoxyphenyl)methyl, and the like. Attachment and cleavageconditions for these and other trityls can be found in Greene and Wuts,Protective Groups in Organic Synthesis, 2nd Edition (John Wiley, NewYork, 1991).

Generally, the nucleotide phosphoramidites of the invention may besynthesized as follows. A nucleoside bearing a hydroxyl protecting groupon the 5'- hydroxyl and a protected complementary functionality on thebase is selectively deprotected to expose only the complementaryfunctionality. Next, a protected dye (as described above) is activatedby converting a linking group into its reactive form. The activatedlinking group of the dye is then reacted with the complementaryfunctionality of the nucleoside to form the dye labeled nucleoside thatbears protecting groups on the 5'-hydroxyl (and on the 2'-hydroxyl forthe case of RNA) and on the phenolic groups of the dye. The dye labelednucleoside is then reacted with a phosphitylating agent as describedabove to produce the nucleotide phosphoramidite.

In a preferred method where the complementary functionality is amine andthe linking group is carboxyl, the synthesis proceeds as follows. Aprotected nucleoside bearing a hydroxyl protecting group on the 5'-hydroxyl, e.g., a trityl group, and a protected amino nitrogencomplementary functionality on the base is selectively deprotected toexpose the amine, such selective deprotection serving to deprotect onlythe amine functionality without deprotecting the protected 5'-hydroxylmoiety. A protected dye (as described above) is activated by convertinga carboxy linking group into its NHS ester with dicyclohexylcarbodiimide and N-hydroxysuccinide. The NHS ester is reacted with theamino group of the nucleoside to form the dye labeled nucleoside thatbears protecting groups on the 5'-hydroxyl (and on the 2'-hydroxyl forthe case of RNA) and on the phenolic groups of the dye. The dye labelednucleoside is then reacted with a phosphitylating agent as describedabove.

C. Polynucleotide Reagents

Yet another preferred class of reagents of the present inventioncomprise polynucleotides labeled with the asymmetric benzoxanthene dyesof the invention. Such labeled polynucleotides are useful in a number ofimportant contexts including as DNA sequencing primers, PCR primers,oligonucleotide hybridization probes, and the like.

As used herein, the terms "polynucleotide" or "oligonucleotide" refer tolinear polymers of natural or modified nucleoside monomers, includingdouble and single stranded deoxyribonucleosides, ribonucleosides,α-anomeric forms thereof and the like. Usually the nucleoside monomersare linked by phosphodiester linkages, where as used herein, the term"phosphodiester linkage" refers to phosphodiester bonds or analogsthereof including phosphorothioate, phosphorodithioate,phosphoroselenoate, phosphorodiselenoate, phosphoroanulothioate,phosphoranilidate, phosphoramidate, and the like, including associatedcounterions, e.g., H, NH₄, Na, and the like if such counterions arepresent. The polynucleotides range in size form a few monomeric units,e.g. 8-40, to several thousands of monomeric units. Whenever apolynucleotide is represented by a sequence of letters, such as"ATGCCTG," it will be understood that the nucleotides are in 5'→3' orderfrom left to right and that "A" denotes deoxyadenosine, "C" denotesdideoxycytidine, "G" denotes deoxyguanosine, and "T" denotes thymidine,unless otherwise noted.

The labeled polynucleotides of the invention include a nucleotide havingthe formula: ##STR23## where B is a 7-deazapurine, purine, or pyrimidinenucleotide base. Z₁ is H or OH. Z₂ is H, OH, HPO₄, or Nuc, wherein Nucrefers to a nucleoside or polynucleotide. The nucleoside of Formula VIand Nuc are linked by a phosphodiester linkage, the linkage beingattached to the 5'-position of Nuc. Z₃ is H, HPO₃, or Nuc, wherein Nucand the nucleoside are linked by a phosphodiester linkage attached tothe 3'-position of Nuc. D is a dye compound of Formula I. Base B isattached to the sugar moiety and to the dye compound as described abovefor the nucleotide phosphoramidite reagent of the invention. As defined,the labeled nucleotide of Formula VI can be the 5'-terminal nucleotide,the 3'-terminal nucleotide, or any internal nucleotide of thepolynucleotide.

In one preferred embodiment, the labeled polynucleotides of the presentinvention include multiple dyes located such that fluorescence energytransfer takes place between a donor dye and an acceptor dye. Suchmulti-dye polynucleotides find application as spectrally-tunablesequencing primers, e.g., Ju et al., Proc. Natl. Acad Sci. USA 92:4347-4351 (1995), and as hybridization probes, e.g., Lee et al. NucleicAcids Research, 21: 3761-3766 (1993).

Labeled polynucleotides may be synthesized either enzymatically, e.g.,using a DNA polymerase or ligase, e.g., Stryer, Biochemisty, Chapter 24,W.H. Freeman and Company (1981), or by chemical synthesis, e.g., by thephosphoramidite method, the phosphite-triester method, and the like,e.g., Gait, Oligonucleotide Synthesis, IRL Press (1990). Labels may beintroduced during enzymatic synthesis utilizing labeled nucleotidetriphosphate monomers as described above, or introduced during chemicalsynthesis using labeled non-nucleotide or nucleotide phosphoramidites asdescribed above, or may be introduced subsequent to synthesis.

Generally, if the labeled polynucleotide is made using enzymaticsynthesis, the following procedure may be used. A template DNA isdenatured and an oligonucleotide primer is annealed to the template DNAA mixture of deoxynucleotide triphosphates is added to the reactionincluding dGTP, dATP, dCTP, and dTTP where at least a fraction of one ofthe deoxynucleotides is labeled with a dye compound of the invention asdescribed above. Next, a polymerase enzyme is added under conditionswhere the polymerase enzyme is active. A labeled polynucleotide isformed by the incorporation of the labeled deoxynucleotides duringpolymerase strand synthesis. In an alternative enzymatic synthesismethod, two primers are used instead of one, one primer complementary tothe + strand and the other complementary to the - strand of the target,the polymerase is a thermostable polymerase, and the reactiontemperature is cycled between a denaturation temperature and anextension temperature, thereby exponentially synthesizing a labeledcomplement to the target sequence by PCR, e.g., PCR Protocols, Innis etal. eds., Academic Press (1990).

Labeled polynucleotides may be chemically synthesized using thephosphoramidite method. Detailed descriptions of the chemistry used toform polynucleotides by the phosphoramidite method are providedelsewhere, e.g., Caruthers et al., U.S. Pat. No. 4,458,066; Caruthers etal., U.S. Pat. No. 4,415,732; Caruthers et al., Genetic Engineering, 4:1-17 (1982); Users Manual Model 392 and 394 Polynucleotide Synthesizers,pages 6-1 through 6-22, Applied Biosystems, Part No. 901237 (1991).Accordingly, each of these references are hereby incorporated byreference in their entirety.

The phosphoramidite method of polynucleotide synthesis is the preferredmethod because of its efficient and rapid coupling and the stability ofthe starting materials. The synthesis is performed with the growingpolynucleotide chain attached to a solid support, so that excessreagents, which are in the liquid phase, can be easily removed byfiltration, thereby eliminating the need for purification steps betweencycles.

The following briefly describes the steps of a typical polynucleotidesynthesis cycle using the phosphoramidite method. First, a solid supportincluding a protected nucleotide monomer is treated with acid, e.g.,trichloroacetic acid, to remove a 5'-hydroxyl protecting group, freeingthe hydroxyl for a subsequent coupling reaction. An activatedintermediate is then formed by simultaneously adding a protectedphosphoramidite nucleoside monomer and a weak acid, e.g., tetrazole, tothe reaction. The weak acid protonates the nitrogen of thephosphoramidite forming a reactive intermediate. Nucleoside addition iscomplete within 30 s. Next, a capping step is performed which terminatesany polynucleotide chains that did not undergo nucleoside addition.Capping is preferably done with acetic anhydride and 1-methylimidazole.The internucleotide linkage is then converted from the phosphite to themore stable phosphotriester by oxidation using iodine as the preferredoxidizing agent and water as the oxygen donor. After oxidation, thehydroxyl protecting group is removed with a protic acid, e.g.,trichloroacetic acid or dichloroacetic acid, and the cycle is repeateduntil chain elongation is complete. After synthesis, the polynucleotidechain is cleaved from the support using a base, e.g., ammonium hydroxideor t-butyl amine. The cleavage reaction also removes any phosphateprotecting groups, e.g., cyanoethyl. Finally, the protecting groups onthe exocyclic amines of the bases and the hydroxyl protecting groups onthe dyes are removed by treating the polynucleotide solution in base atan elevated temperature, e.g., 55° C.

Any of the phosphoramidite nucleoside monomers may be dye-labeledphosphoramidites as described above. If the 5'-terminal position of thenucleotide is labeled, a labeled non-nucleotidic phosphoramidite of theinvention may be used during the final condensation step. If an internalposition of the oligonucleotide is to be labeled, a labeled nucleotidicphosphoramidite of the invention may be used during any of thecondensation steps.

Subsequent to synthesis, the polynucleotide may be labeled at a numberof positions including the 5'-terminus, e.g., Oligonucleotides andAnalogs, Eckstein ed., Chapter 8, IRL Press (1991) and Orgel et al.,Nucleic Acids Research 11(18): 6513 (1983); U.S. Pat. No. 5,118,800,hereby incorporated by reference; the phosphodiester backbone, e.g.,ibid., Chapter 9; or at the 3'-terminus, e.g., Nelson, Nucleic AcidsResearch 20(23): 6253-6259, and U.S. Pat. Nos. 5,401,837 and 5,141,813,both patents hereby incorporated by reference. For a review ofoligonucleotide labeling procedures see R. Haugland in Excited States ofBiopolymers, Steiner ed., Plenum Press, NY (1983).

In one preferred post-synthesis chemical labeling method anoligonucleotide is labeled as follows. A dye including a carboxy linkinggroup is converted to the n-hydroxysuccinimide ester by reacting withapproximately 1 equivalent of 1,3-dicyclohexylcarbodiimide andapproximately 3 equivalents of n-hydroxysuccinimide in dry ethyl acetatefor 3 hours at room temperature. The reaction mixture is washed with 5%HCl, dried over magnesium sulfate, filtered, and concentrated to a solidwhich is resuspended in DMSO. The DMSO dye stock is then added in excess(10-20×) to an aminohexyl derivatized oligonucleotide in 0.25 Mbicarbonate/carbonate buffer at pH 9.4 and allowed to react for 6 hours,e.g., U.S. Pat. No. 4,757,141. The dye labeled oligonucleotide isseparated from unreacted dye by passage through a size-exclusionchromatography column eluting with buffer, e.g., 0.1 molar triethylamineacetate (TEAA). The fraction containing the crude labeledoligonucleotide is further purified by reverse phase HPLC employinggradient elution.

IV. Methods Utilizing the Compounds and Reagents of the Invention

The dyes and reagents of the present invention are well suited to anymethod utilizing fluorescent detection, particularly methods requiringthe simultaneous detection of multiple spatially-overlapping analytes.Dyes and reagents of the invention are particularly well suited foridentifying classes of polynucleotides that have been subjected to abiochemical separation procedure, such as electrophoresis, where aseries of bands or spots of target substances having similarphysiochemical properties, e.g. size, conformation, charge,hydrophobicity, or the like, are present in a linear or planararrangement. As used herein, the term "bands" includes any spatialgrouping or aggregation of analytes on the basis of similar or identicalphysiochemical properties. Usually bands arise in the separation ofdye-polynucleotide conjugates by electrophoresis.

Classes of polynucleotides can arise in a variety of contexts. In apreferred category of methods referred to herein as "fragment analysis"or "genetic analysis" methods, labeled polynucleotide fragments aregenerated through template-directed enzymatic synthesis using labeledprimers or nucleotides, e.g., by ligation or polymerase-directed primerextension; the fragments are subjected to a size-dependent separationprocess, e.g., electrophoresis or chromatography, and, the separatedfragments are detected subsequent to the separation, e.g., bylaser-induced fluorescence. In a particularly preferred embodiment,multiple classes of polynucleotides are separated simultaneously and thedifferent classes are distinguished by spectrally resolvable labels.

One such fragment analysis method known as amplified fragment lengthpolymorphisim detection (AmpFLP) is based on amplified fragment lengthpolymorphisms, i.e., restriction fragment length polymorphisms that areamplified by PCR. These amplified fragments of varying size serve aslinked markers for following mutant genes through families. The closerthe amplified fragment is to the mutant gene on the chromosome, thehigher the linkage correlation. Because genes for many genetic disordershave not been identified, these linkage markers serve to help evaluatedisease risk or paternity. In the AmpFLPs technique, the polynucleotidesmay be labeled by using a labeled polynucleotide PCR primer, or byutilizing labeled nucleotide triphosphates in the PCR.

In another such fragment analysis method known as nick translation, areaction is used to replace unlabeled nucleoside triphosphates in adouble-stranded DNA molecule with labeled ones. Free 3'-hydroxyl groupsare created within the unlabeled DNA by "nicks" caused bydeoxyribonuclease I (DNAase I) treatment. DNA polymerase I thencatalyzes the addition of a labeled nucleotide to the 3'-hydroxylterminus of the nick. At the same time, the 5' to 3'-exonucleaseactivity of this enzyme eliminates the nucleotide unit from the5'-phosphoryl terminus of the nick. A new nucleotide with a free 3'-OHgroup is incorporated at the position of the original excisednucleotide, and the nick is shifted along by one nucleotide unit in the3' direction. This 3' shift will result in the sequential addition ofnew labeled nucleotides to the DNA with the removal of existingunlabeled nucleotides. The nick-translated polynucleotide is thenanalyzed using a separation process, e.g., electrophoresis.

Another exemplary fragment analysis method is based on variable numberof tandem repeats, or VNTRs. VNTRs are regions of double-stranded DNAthat contain adjacent multiple copies of a particular sequence, with thenumber of repeating units being variable. Examples of VNTR loci arepYNZ22, pMCT118, and Apo B. A subset of VNTR methods are those methodsbased on the detection of microsatellite repeats, or short tandemrepeats (STRs), i.e., tandem repeats of DNA characterized by a short (24bases) repeated sequence. One of the most abundant interspersedrepetitive DNA families in humans is the (dC-dA)n-(dG-dT)n dinucleotiderepeat family (also called the (CA)n dinucleotide repeat family). Thereare thought to be as many as 50,000 to 100,000 (CA)n repeat regions inthe human genome, typically with 15-30 repeats per block Many of theserepeat regions are polymorphic in length and can therefore serve asuseful genetic markers. Preferably, in VNTR or STR methods, label isintroduced into the polynucleotide fragments by using a dye-labeled PCRprimer.

In a particularly preferred fragment analysis method, classes identifiedin accordance with the invention are defined in terms of terminalnucleotides so that a correspondence is established between the fourpossible terminal bases and the members of a set of spectrallyresolvable dyes. Such sets are readily assembled from the dyes of theinvention by measuring emission and absorption bandwidths withcommercially available spectrophotometers. More preferably, the classesarise in the context of the chemical or chain termination methods of DNAsequencing, and most preferably the classes arise in the context of thechain termination method, i.e., dideoxy DNA sequencing, or Sangersequencing. This method involves the synthesis of a DNA strand by a DNApolymerase in vitro using a single-stranded or double-stranded DNAtemplate whose sequence is to be determined. Synthesis is initiated atonly the one site where an oligonucleotide primer anneals to thetemplate. The synthesis reaction is terminated by incorporation of anucleotide analog that will not support continued DNA elongation. Thechain-terminating nucleotide analogs are the 2',3'-dideoxynucleoside5'-triphosphates (ddNTPs) which lack the 3'-OH group necessary for 3' to5' DNA chain elongation. When proper proportions of ddNTPs(2'-deoxynucleoside 5'-triphosphates) and one of the four ddNTPs areused, enzyme-catalyzed polymerization will be terminated in a fractionof the population of chains at each site where the ddNTP can beincorporated. If labeled primers or labeled ddNTPs are used for eachreaction, the sequence information can be detected by fluorescence afterseparation by high-resolution electrophoresis. In the chain terminationmethod, dyes of the invention can be attached to either sequencingprimers or dideoxynucleotides. Dyes can be linked to a complementaryfunctionality on the 5' end of the primer, e.g. following the teachingin Fung et al, U.S. Pat. No. 4,757,141 which is incorporated herein byreference; on the base of a primer, or on the base of adideoxynucleotide, e.g. via the alkynylamino linking groups disclosed byHobbs et al, European patent application number 87305844.0 which isdiscussed above and incorporated herein by reference.

In each of the above fragment analysis methods labeled polynucleotidesare preferably separated by electrophoretic procedures, e.g. Gould andMatthews, cited above; Rickwood and Hames, Eds., Gel Electrophoresis ofNucleic Acids: A Practical Approach, (IRL Press Limited, London, 1981);or Osterman, Methods of Protein and Nucleic Acid Research, Vol. 1Springer-Verlag, Berlin, 1984). Preferably the type of electrophoreticmatrix is crosslinked or uncrosslinked polyacrylamide having aconcentration (weight to volume) of between about 2-20 weight percent.More preferably, the polyacrylamide concentration is between about 48percent. Preferably in the context of DNA sequencing in particular, theelectrophoresis matrix includes a strand separating, or denaturing,agent, e.g., urea, formamide, and the like. Detailed procedures forconstructing such matrices are given by Maniatis et al., "Fractionationof Low Molecular Weight DNA and RNA in Polyacrylamide Gels Containing98% Formamide or 7 M Urea," in Methods in Enymology, 65: 299-305 (1980);Maniatis et al., "Chain Length Determination of Small Double- andSingle-Stranded DNA Molecules by Polyacrylamide Gel Electrophoresis,"Biochemisty, 14: 3787-3794 (1975); Maniatis et al., Molecular Cloning: ALaboratory Manual (Cold Spring Harbor Laboratory, New York, 1982), pgs.179-185; and ABI PRISM™ 377 DNA Sequencer User's Manual, Rev. A, January1995, Chapter 2 (p/n 903433, The Perkin-Elmer Corporation, Foster City,Calif.). Accordingly these references are incorporated by reference. Theoptimal polymer concentration, pH temperature, concentration ofdenaturing agent, etc. employed in a particular separation depends onmany factors, including the size range of the nucleic acids to beseparated, their base compositions, whether they are single stranded ordouble stranded, and the nature of the classes for which information issought by electrophoresis. Accordingly application of the invention mayrequire standard preliminary testing to optimize conditions forparticular separations. By way of example, oligonucleotides having sizesin the range of between about 20-300 bases have been separated anddetected in accordance with the invention in the following matrix: 6percent polyacrylamide made from 19 parts to 1 part acrylamide tobis-acrylamide, formed in a Tris-borate EDTA buffer at pH 8.3.

Subsequent to electrophoretic separation, the dye-polynucleotideconjugates are detected by measuring the fluorescence emission from thedye labeled polynucleotides. To perform such detection, the labeledpolynucleotides are illuminated by standard means, e.g. high intensitymercury vapor lamps, lasers, or the like. Preferably the illuminationmeans is a laser having an illumination beam at a wavelength between 488and 550 nm. More preferably, the dye-polynucleotides are illuminated bylaser light generated by an argon ion laser, particularly the 488 and514 nm emission lines of an argon ion laser, or an the 532 emission lineof a neodymium solid-state YAG laser. Several argon ion lasers areavailable commercially which lase simultaneously at these lines, e.g.Cyonics, Ltd. (Sunnyvale, Calif.) Model 2001, or the like. Thefluorescence is then detected by a light-sensitive detector, e.g., aphotomultiplier tube, a charged coupled device, or the like.

IV. Examples

The invention will be further clarified by a consideration of thefollowing examples, which are intended to be purely exemplary of theinvention and not to in any way limit its scope.

Unless otherwise indicated, all chemicals were obtained from AldrichChemical Company (Milwaukee, Wis.) and used as purchased.3-Fluororesorcinol (11a) was synthesized from 2,4-dimethoxyanilineaccording to the literature procedure (Perkin, J. Chem Soc. 110:1658-1666 (1980)). 2-Chloro-4-methoxyresorcinol (11c) was synthesizedfrom 3-hydroxy-4-methoxy-benzaldehyde according to U.S. Pat. No.4,318,846. 3,6-Dichlorotrimelletic acid was synthesized according toU.S. Pat. No. 4,318,846, and converted to the anhydride 10a by refluxingin neat acetic anhydride for 4 hours and precipitation of the cooledmixture with diethyl ether. Ethyl hydrogen fluoromalonate wassynthesized from diethyl fluoromalonate according to the literature(Org. Syn. Coll. 4: 417-419 (1963)). Ethyltributylphosphonium-fluoroacetate (19) was synthesized according to theliterature (Tet Lett. 30: 6113 (1980)). 2-Fluoro-1,3-dihydroxynapthalene(9a) was synthesized as described in the present disclosure. Drydichloromethane (CH₂ Cl₂) was distilled from calcium hydride andtetrahydrofuran (THF) from lithium aluminum hydride (LAH) prior to use.Absolute ethanol was used as purchased or dried by distillation fromsodium and stored over activated molecular sieves. Dry ethyl acetate(EtOAc) was distilled from P₂ O₅ after predrying with MgSO₄. Drydimethylformamide (DMF) was distilled after predrying with magnesiumsulfate and stored over activated molecular sieves. All reactions wererun under anhydrous conditions under dry argon. Reactions were monitoredby thin layer chromatography (TLC) (Silica gel 60, A₂₅₄). Flashchromatography was performed on silica gel 60 (200-400 mesh, Baxter).Final purification of the asymmetric benzoxanthene dyes to give the pureisomers, designated "1" and "2", employed preparative TLC on silica gel60 PTLC plates (EM Science) eluting with CH₂ Cl₂ :MEOH:AcOH (7:3:0.1).Pure dye isomers were identified by giving a single spot on TLCemploying CH₂ Cl₂ :MEOH:AcOH (7:3:0.1) and visualizing with short andlong-wavelength UV irradiation. Isomer 2 runs slower on both normal andreverse-phase media. Intermediate products were identified by ¹ HNMRspectra on a Varian 300 MHz NMR. Absorption spectra of the purified dyeswere recorded on a Hewlett Packard 8451A diode array spectrophotometer,and fluorescence emission spectra were recorded on a Perkin Elmer LS50-B luminescence spectrophotometer. HPLC purification of dye labeledoligonucleotides was performed on a Perkin-Elmer 200 series pump,connected to a PE LC 240 fluorescence detector, and a PE LC 295 UV/VISdetector, connected to a 2 channel PE 1022 integrator. Buffers employedfor dye labeled oligonucleotide purification and identification includetris(hydroxymethyl) aminomethane / borate / EDTA (TBE),tris(hydroxymethyl) aminomethane / EDTA (TE), triethylammonium acetate(TEAA). Buffers are stored as 10×solutions at 0° C. and diluted freshbefore use. HPLC purification employed a reverse-phase RP-18 column.

EXAMPLE 1 Synthesis of Asymmetric Benzoxanthenes

Compounds 1-7 in FIGS. 2A and 2B were synthesized by reacting a 1,3dihydroxynapthalene derivative, such as 1,3-dihydroxynapthalene 9b or2-fluoro-1,3-dihydroxynapthalene 9a (0.2 mole), with 1.1 equivalent ofthe phthallic anhydride derivative 3,6dichlorotrimelletic acid anhydride10a, and one equivalent of a resorcinol derive 11 (0.2 mole), 11a, 11b,11c, or 11d depending on the final product desired, and heated for 16hours in neat MeSO₃ H (3 ml) at 110° C. under Argon. The crude dye (amixture of regioisomers in reactions employing 10a) was precipitated byaddition to an ice / water mixture and isolated by filtration. The crudedye was purified into 2 isomers 1 and 2 by preparative thin layerchromatography eluting with a mixture of CH₂ Cl₂ :MEOH:Acetic Acid(70:30:1).

The inset in FIG. 2B shows that R₂ and/or R₃ unsubstituted (R₂ =R₃ =H)derivatives of the asymmetric benzoxanthene dyes, shown for isomer 2 ofdye 5, react further with halogenating reagents (NaOCl, NaOH / Br₂, NaOH/ I₂) at 0° C. for 3 hours to produce quantitatively the halogenatedderivatives such as 8 (R2=R3=Cl, Br, I, F) after extractive workup with10% HCl / EtOAc, drying with Na₂ SO₄, filtering, and concentrating invacuo.

EXAMPLE 2 Synthesis of Dye-labeled Oligonucleotides

The synthesis of dye labeled oligonucleotides of the invention will bedescribed with reference to FIG. 3. Cl-FLAN, dye 2, was converted to then-hydroxysuccinimide ester 12 by reacting with 1.2 equivalents of1,3-dicyclohexylcarbodiimide and 3 equivalents of n-hydroxysuccinimidein dry ethyl acetate for 3 hours at room temperature. The reactionmixture was washed with 5% HCl, dried over magnesium sulfate, filtered,and concentrated to a solid which was resuspended in DMSO (10 mg dye /50 μL DMSO). The DMSO dye stock (5-10 μg) was added in excess (10-20×)to an aminohexyl derivatized -21M13 oligonucleotide primer (1×10⁻³ M) in0.25 M bicarbonate/carbonate buffer at pH 9.4 and allowed to react for 6hours. The aminohexyl derivatized primer was prepared by automatedsolid-phase DNA synthesis using Aminolink-2 in the last cycle (PE p/n400808). The dye labeled oligonucleotide was separated from unreacteddye by passage through a Sephadex G-25 column eluting with 0.1 molartriethylamine acetate (TEAA). The fraction containing the crude labeledoligonucleotide was purified by reverse phase HPLC employing gradientelution from 8% AcCN in 0.1 M TEAA to 25% over 25 minutes using an RP-18chromatography column. The pure dye labeled oligonucleotide 13 waslyophilized to a solid and resuspended in 1×TE buffer pH 8.4. Theconcentration of the dye labeled oligonucleotide was determined by UVabsorption at 260 nm assuming additive extinction coefficient values of6,650 for T, 7,350 for C, 11,750 for G, and 14,900 for A, and therelative contribution of the dye absorption at 260 nm determined fromspectra of the free dye measured in the same buffer.

EXAMPLE 3 Comparison of the Excitation Spectra of TAMRA (22) and Cl-FLAN(2) Labeled Oligonucleotides from Example 2

Excitation spectra were recorded for each dye in 1×TBE buffer at pH 8.4.Dyes where present at an equimolar concentration (ca. 1×10⁻⁶ M). Theemission intensity was recorded at λ_(max) Em for each dye. FIG. 4 showsthat for excitation at 488 nm the relative excitation efficiency ofCl-FLAN is approximately 2.5 times that of the TAMRA dye, while forexcitation at 514 nm, the relative excitation efficiency of Cl-FLAN isapproximately 1.5 times that of the TAMRA dye.

EXAMPLE 4 Comparison of the Quantum Yield of TAMRA (22) and Cl-FLAN (2)Labeled Oligonucleotides from Example 2

FIG. 5 shows emission spectra the fluorescence emission intensity of aTAMRA (22) labeled -21M13 oligonucleotide and a Cl-FLAN(2) labeled-21M13 oligonucleotide excited at the absorption maxima of each dye. Theoligonucleotides were prepared as in Example 2. The data demonstrate a60% greater quantum yield for the Cl-FLAN (2) labeled oligonucleotide ascompared to the TAMRA (22) labeled oligonucleotide. Spectra wererecorded in 1×TE buffer at pH 8.4 at a concentration resulting in anequal λ_(max) Abs of 0.05 for each labeled oligonucleotide. Emissionspectra were recorded for each dye with irradiation at λ_(max) Abs foreach dye.

EXAMPLE 5 Comparison of the Molar Emission Intensity of Cl-FLAN (2) andTAMRA (22) Labeled Oligonucleotides

Emission spectra of equimolar concentrations (ca. 1×10⁻⁶ M) of a TAMRA(22) labeled oligonucleotide and a Cl-FLAN (2) labeled oligonucleotidedissolved in 1×TE buffer at pH 8.4 were measured by irradiating eacholigonucleotide at 488 nm and 514 nm, and adding the spectra toapproximate the radiation of a multiline argon laser. FIG. 6 shows thatthe fluorescence intensity of the Cl-FLAN (2) labeled oligonucleotide isover 2 times greater than that of the TAMRA (22) labeledoligonucleotide.

EXAMPLE 6 Multiplex Dye-labeled Oligonucleotide Set

Long-wavelength fluorescence emission of a Cl-FLAN (2) labeledoligonucleotide -21M13 sequencing primer was compared with the emissionfrom -21M13 sequencing primers labeled with 6-FA, TET, and, HEX 23 dyes,where 6-FAM refers to 6-carboxyfluorescein, "TET" refers to6-carboxy-4,7,2',7'-tetrachlorofluorescein, and "HEX" refers to6-carboxy-4,7,2',4',5',7'-hexachlorofluorescein. Primers were labeled asdescribed above in Example 2. The excitation wavelength was 490 nm.Emission spectra were run in 1×TE buffer at pH 8.4 and normalized toequal intensity (ca. 1*10⁻⁶ M). FIG. 7 shows that the 573 nm emissionmaxima and the narrow width of the emission spectrum of the Cl-FLAN (2)labeled oligonucleotide makes the Cl-FLAN (2) labeled oligonucleotidespectrally resolved from the emission spectra of the other 3 dyes in theset. Such spectral resolution indicates the suitability of a dye setincluding, FAM, TET, and HEX labeled oligonucleotides with the Cl-FLAN(2) asymmetric benzoxanthene dye.

EXAMPLE 7 Synthesis of a 2-Fluoro-1,3-Dihydroxynapthalene Intermediate

See FIG. 8. Commercially available homopthallic anhydride (14) (100 gm)was reacted with ethanol (300 mL) under acid catalysis (0.5 mL TFA) toproduce a 95% yield of the intermediate ethyl ester 15 after refluxingfor 3 hours, concentration to a solid, and recrystallization fromtoluene. Intermediate 15 (10 gm) was then reacted with 1.1 equivalentsof oxalyl chloride in CH₂ Cl₂ (200 mL) for 4 hours at room temperatureto produce an 80% yield of acid chloride 16 as a crude solid afterconcentration at room temperature under high vacuum. Crude 16 wassuspended in THF and reacted by either of the following two methods withfluoro acetate equivalents to produce compound 20.

Method A: The potassium salt (17) of ethyl fluoroacetate (3equivalents), formed by reaction of ethyl fluoroacetate and potassiumt-butoxide at 0° C. in THE, or the magnesium salt of ethyl hydrogenfluoromalonate (18) (1.5 equivalents), formed by reaction of isopropylmagnesium bromide (2 equivalents) and ethyl hydrogen fluoromalonate at-60° C., were added slowly to the THF suspension of 16 and allowed toreact for 6 hours at 0° C. The reaction was quenched by adding 5% HCl,extracted (3 times) with EtOAc, the organic layer was dried,concentrated, and the resulting crude mixture purified by flashchromatography employing gradient elution from 6:4 hexanes/CH₂ Cl₂ to100% CH₂ Cl₂ giving 35 to 50% yield of compound 20.

Method B: The phosphorous yield 19 was slowly added to the THFsuspension of 16 at -70° C., then allowed to warm to room temperatureand react for 16 hours. The reaction was quenched by addition of 5%NaHCO₃ and stirred for 6 hours. The reaction was extracted with THF /water (3 times) and the product was isolated as for Method A to produceintermediate 20 in >50% yield. Purified 20 intra-molecularly cyclizedunder base catalysis (2 equivalents NaOEt) to a cyclic intermediate 21which decarboxylated in situ to give the2-fluoro-1,3-dihydroxynapthalene (9a) in 50% yield. Alternatively, thecyclic intermediate 21 can be isolated in >80% yield when employingpotassium t-butoxide in THF and decarboxylkated to2-fluoro-1,3-dihydroxynapthalene (9a).

EXAMPLE 8 DNA Sequencing Employing Asymmetric Benzoxanthene Compound 2

Automated cycle sequencing was performed using a Perkin-Elmer Catalyst800 Molecular Biology Labstation (The Perkin-Elmer Corporation, FosterCity, Calif. (PE)). Four separate Sanger sequencing reactions were runemploying the same -21 M13 primer labeled with 6-FAM (C terminator), TET(A terminator), HEX (G terminator), or Cl-FLAN 2 (T terminator) asdescribed below. A mixture of the four reactions was loaded and data wasgenerated on a Perkin-Elmer ABI Prism™ 377 DNA sequencer and associateddata analysis software.

Cycle sequencing reactions were performed on the Catalyst 800 MolecularBiology Labstation using the 3.02 platform software. The Catalyst wasprogrammed to deliver 0.6 μL of pGEM 3Z+ template DNA at a concentrationof 100 ng/μL, and 1.9 μL of premix defined below. Sequencing data wasgenerated on an ABI Prism™ 377 DNA Sequencer using a 5% Long Ranger gel(FMC corporation, Rockland, Me.). Each of the four sequencing premixesis defined below in Table I:

                  TABLE I                                                         ______________________________________                                        A Premix                                                                             60 mM Tris pH 9.0; 2.5 mM MgCl2; 4 mM Kcl;                                0.04 mM DTT; 4 μMEDTA; 0.1 μM TET labeled primer;                       0.66 U/μL Amplitaq FS; 1.66 U/μL rTth Pyrophosphatase;                  0.5 μM ddATP; 125 μM dATP; 125 μM dCTP;                              150 μM c7dGTP; 125 μM dTTP.                                            C Premix 60 mM Tris pH 9.0; 2.5 mM MgCl2; 4 mM KCl;                            0.04 mM DTT; 4 μM EDTA; 0.1 μM 6-FAM labeled primer;                    0.66 U/μL Amplitaq FS; 1.66 U/μL rTth                                   Pyrophosphatase; 0.5 μM ddCTP; 125 μM dATP; 125 μM                   dCTP; 150 μM c7dGTP; 125 μM dTTP.                                      G Premix 60 mM Tris pH 9.0; 2.5 mM MgCl.sub.2, 4 mM Kcl;                       0.04 mM DTT; 4 μM EDTA; 0.1 μM HEX                                      labeled primer; 0.66 U/μL Amplitaq FS; 1.66                                U/μL rTth Pyrophosphatase; 0.375 μM ddGTP;                               125 μM dATP; 125 μM dCTP; 150 μM c7dGTP;                            125 μM dTTP.                                                              T Premix 60 mM Tris pH 9.0; 2.5 mM MgCl2; 4 mM Kcl;                            0.04 mM DTT; 4 μM EDTA; 0.1 μM FLAN labeled primer;                     0.66 U/μL Amplitaq FS; 1.66 U/μL rTth                                   Pyrophosphatase; 0.875 μM ddTTP; 125 μM dATP; 125                       μM dCTP; 150 μM c7dGTP; 125 μM dTTP.                              ______________________________________                                    

Cycle sequencing was performed on the above mixtures of template andpremixes. The cycling conditions on the Catalyst were as follows: onecycle of 96° C. for 20 seconds; 15 cycles of 94° C. for 20 seconds, 55°C. for 40 seconds, and 68° C. for 60 seconds; and 15 cycles of 94° C.for 20 seconds and 68° C. for 60 seconds.

Following thermal cycling, the four separate reactions were combinedinto the concentration buffer (83% DMSO/25 mM EDTA / 8 mg/ml BlueDextran ) and concentrated using standard Express Load methods (v 2.02Catalyst Manual, PE). 2 mL of concentrated sample was loaded onto a wellof the 377 sequencer, run, and analyzed using version 1.1 Software. Thesequence between base 233 and 263 is shown in FIG. 9.

EXAMPLE 9 Microsatellite Fragments Labeled using Cl-FLAN (2), HEX andTET Labeled Primers Separated Simultaneously with ROX Labeled InternalSize Standards

PCR reactions of four loci of a human CEPH family DNA using dye labeledprimers was performed as described below. The PCR products were pooledand electrophoretically separated on a Perkin-Elmer ABI Prism 377™ DNAsequencer (PE). The unique fluorescent signal of each dye labeledfragment peak was analyzed using GeneScan™ Analysis Software v 2.0.2(PE). Referring to FIG. 10, the red peaks (labeled R) correspond to ROX(26) labeled internal standard fragments, the blue peaks (labeled B)correspond to TET labeled fragments, the green peaks (labeled G)correspond to HEX labeled fragments, and the black peaks (labeled K)correspond to Cl-FLAN (2) labeled fragments.

The PCR reactions were run on a Perkin-Elmer 9600 thermocycler (PE). Aseparate reaction was performed for each dye labeled primer employingthe following cocktail:

    ______________________________________                                        Reaction Components Volume (μL)                                            ______________________________________                                        Dye labeled Primer Mix (5 μM)                                                                  1.0                                                         DNA (50 ng/μL) 1.2                                                         10X PE PCR Buffer II 1.5                                                      dNTP mix (2.5 mM) 1.5                                                         AmpliTaq.sup.a (5 units/μL) 0.12                                           2.0 mM MgCl.sub.2 1.2                                                         Sterile D.I. Water 8.48                                                       Total Mix 15.0                                                              ______________________________________                                    

The mixtures were amplified using the following cycling conditions: 1cycle at 95° C. for 5 minutes; 10 cycles at 94° C. for 15 seconds, 55°C. for 15 seconds, and 72° C. for 30 seconds; 20 cycles at 89° C. for 15seconds, 55° C. for 15 seconds, and 72° C. for 30 seconds; and 1 cycleat 72° C. for 10 minutes.

The amplified PCR Products were pooled by mixing the Cl-FLAN (2) andTET-labeled PCR products (0.5 μL) with 1.0 μL of each HEX labeled PCRproduct to give an overall ratio of mixed dye labeled fragmentsconsisting of 1:2:1 (Cl-FLAN:HEX:TET). The pooled PCR fragments weremixed with a loading cocktail consisting of 2.5 μL formamide, 0.5 μLBlue Dextran (50 mM EDTA, 50 mg/mL Blue Dextran), and 0.5 μL SizeStandard (GS-350 ROX PE p/n 401735). The pooled mixture was denatured at95° C. for five minutes and then loaded onto one gel lane of a PE ABIPrism™ 377 DNA sequencer. The fragments were electrophoreticallyseparated and detected using an acrylamide gel having the followingcharacteristics: 0.20 mm thickness, 4.25% (wt) acrylamide, 19:1acrylamide/bisacrylamide (wt/wt), 34-well square tooth comb, 10× TBEBuffer (89 mM Tris, 89 mM Boric Acid, 2 mM EDTA) pH of 8.3. Theinstrument was run using Filter Wheel A and the GS 36D-2400 Module whichhas the following run parameters: EP Voltage of 3000 V, EP Current of60.0 mA, EP Power of 200 W, Gel Temperature of 51° C. and a laser powerof 40 mW.

EXAMPLE 10 Comparison of the Spectral Properties, Photostability, andChemical Stability of Rhodamine Dyes, Xanthene Dyes and the AsymmetricXanthene Dyes of the Invention

Table I below summarizes and compares various spectral and chemicalproperties of the asymmetric benzoxanthene dyes of the invention andother spectrally similar xanthene and rhodamine-based dyes.

                  TABLE II                                                        ______________________________________                                                        Width at  Relative     Stability in                              .sub.max Em Half Height Photo- Relative NH.sub.4 OH-t.sub.1/2                                                      Dye (nm) (nm) stability Brightness                                            (hr)                                  ______________________________________                                        HEX     550     32        17    2.4     11                                      (5) 552 45 2.8 3.9 430                                                        (6) 554 47 -- -- --                                                           (4) 564 41 -- -- --                                                           (1) 565 45 5.3 1.6 478                                                        (2) 568 42 5.1 2.1 146                                                        CL-FLAN                                                                       (7) 570 45 1.1 1.6 --                                                         (8) 572 47 -- -- --                                                           TAMRA 577 39 -- 0.9    1.3                                                    NAN 579 44 0.3 1    52                                                        (3) 583 43 1.7 1.1  14                                                        ROX 594 53 -- 0.5 272                                                         DEB 598 48 0.3 0.3 --                                                       ______________________________________                                    

See FIGS. 1 and 2 for the structures of the dyes referred to in thetable. All data are reported for pure dye isomer 2. All emission spectrawere recorded in 1×TBE buffer at pH 8.4 in dye solutions having anabsorbance of 0.05 at λ_(max) Abs (ca. 1×10⁻⁶ M) at room temperature.Photodecomposition rate was determined for equal volumes of the dyes atinitially 1 absorption unit at λ_(max) Abs and run in pairs at equalvolumes under equal high intensity white light irradiation at 35° C. in1×TBE buffer pH 8.4. Absorption spectra of aliquots were taken at 1 hourintervals and the intensities at λ_(max) Em were fitted with first orderexponential curves to determine the t_(1/2) rate for loss of dye.Relative brightness at λ_(max) Em was determined using 514 nm excitationof dyes at approximately equal concentrations. (λ_(max) Abs=0.05). Forthe NH₄ OH stability measurements the dyes were diluted in concentratedammonia hydroxide at approximately equal concentrations (λ_(max) Abs=1)and incubated at 60° C. for 20 hours in sealed vials. Absorption spectraof aliquots were taken at 1 hour intervals and the intensities atλ_(max) Em were fitted with first order exponential curves to determinethe t_(1/2) for dye decomposition.

All publications and patent applications are herein incorporated byreference to the same extent as if each individual publication or patentapplication was specifically and individually indicated to beincorporated by reference.

Those having ordinary skill in the chemical and biochemical arts willclearly understand that many modifications are possible in the preferredembodiment without departing from the teachings thereof all suchmodifications are intended to be encompassed within the followingclaims.

We claim:
 1. An asymmetric benzoxanthene dye compound having theformula: ##STR24## wherein: Y₁ and Y₂ taken separately are selected fromthe group consisting of hydroxyl, oxygen, imminium, and amine;R₁ -R₈taken separately are selected from the group consisting of hydrogen,fluorine, chlorine, lower alkyl, lower alkene, lower alkyne, sulfonate,sulfone, amino, imminium, amido, nitrile, lower alkoxy, linking group,and combinations thereof; and R₉ is selected from the group consistingof acetylene, lower alkyl, lower alkene, cyano, phenyl, heterocyclicaromatic, and substituted phenyl having the structure: ##STR25##wherein: X₁ -X₅ taken separately are hydrogen, chlorine, fluorine, loweralkyl, carboxylic acid, sulfonic acid, --CH₂ OH, or linking group. 2.The compound of claim 1 wherein one of Y₁ and Y₂ is oxygen and the otheris hydroxyl.
 3. The compound of claim 1 wherein:X₁ is selected from thegroup consisting of carboxylic acid, sulfonic acid, and --CH₂ OH; X₂ andX₅ taken separately are selected from the group consisting of hydrogen,chlorine, fluorine, and lower alkyl; and X₃ and X₄ taken separately areselected from the group consisting of hydrogen, chlorine, fluorine,lower alkyl, carboxylic acid, sulfonic acid, and linking group.
 4. Thecompound of claim 1 wherein X₂ and X₅ are chlorine.
 5. The compound ofclaim 1 wherein X₁ is carboxylic acid.
 6. The compound of claim 1wherein one of X₃ or X₄ is linking group, the other being hydrogen. 7.The compound of claim 1 wherein one of X₁ or X₅ is selected from thegroup consisting of carboxylic acid, sulfonic acid, and --CH₂ OH.
 8. Thecompound of claim 1 wherein one of R₁ -R₃ is fluorine.
 9. The compoundof claim 8 wherein R₃ is fluorine.
 10. The compound of claim 1wherein:one of Y₁ and Y₂ is oxygen and the other is hydroxyl; R₁ is achlorine; R₃ is a fluorine; R₂ and R₄ -R₈ are hydrogen; and R₉ issubstituted phenyl wherein X₁ is carboxyl, X₂ and X₅ are chlorine, andone of X₃ and X₄ is carboxyl and the other is hydrogen.
 11. The compoundof claim 1 wherein:one of Y₁ and Y₂ is oxygen and the other is hydroxyl;R₁ and R₃ are fluorine; R₂, and R₄ -R₈ are hydrogen; and R₉ issubstituted phenyl wherein X₁ is carboxyl, X₂ and X₅ are chlorine, andone of X₃ and X₄ is carboxyl and the other is hydrogen.
 12. The compoundof claim 1 wherein:one of Y₁ and Y₂ is oxygen and the other is hydroxyl;R₁ is methoxy, R₂ is chlorine, R₃ is fluorine; R₄ -R₈ are hydrogen; andR₉ is substituted phenyl wherein X₁ is carboxyl, X₂ and X₅ are chlorine,and one of X₃ and X₄ is carboxyl and the other is hydrogen.
 13. Thecompound of claim 1 wherein:one of Y₁ and Y₂ is oxygen and the other ishydroxyl; R₃ is fluorine; R₁, R₂, and R₄ -R₈ are hydrogen; and R₉ issubstituted phenyl wherein X₁ is carboxyl, X₂ and X₅ are chlorine, andone of X₃ and X₄ is carboxyl and the other is hydrogen.
 14. The compoundof claim 1 wherein:one of Y₁ and Y₂ is oxygen and the other is hydroxyl;R₁ -R₈ are hydrogen; and R₉ is substituted phenyl wherein X₁ is carboxylX₂ and X₅ are chlorine, and one of X₃ and X₄ is carboxyl and the otheris hydrogen.
 15. The compound of claim 1 wherein:one of Y₁ and Y₂ isoxygen and the other is hydroxyl; R₁ is chlorine; R₂ -R₈ are hydrogen;and R₉ is substituted phenyl wherein X₁ is carboxyl, X₂ and X₅ arechlorine, and one of X₃ and X₄ is carboxyl and the other is hydrogen.16. The compound of claim 1 wherein:one of Y₁ and Y₂ is oxygen and theother is hydroxyl; R₁ is methoxy; R₂ is chlorine; R₃ -R₈ are hydrogen;and R₉ is substituted phenyl wherein X₁ is carboxyl X₂ and X₅ arechlorine, and one of X₃ and X₄ is carboxyl and the other is hydrogen.17. A phosphoramidite compound having the formula: ##STR26## wherein: Xis a spacer arm;Y is a linkage; B₁ is a phosphite ester protectinggroup; B₂, and B₃ taken separately are selected from the groupconsisting of lower alkyl, lower alkene, aryl, and cycloalkyl containingup to 10 carbon atoms; and D is the dye compound of claim 1; wherein Yand D are linked through a linkage attached to dye D at one of positionsR₁ -R₉.
 18. The compound of claim 17 wherein B₂ and B₃ taken togetherform an alkene chain containing up to 5 carbon atoms in the principlechain and a total of up to 10 carbon atoms with both terminal valencebonds of said chains being attached to the nitrogen atom; or B₂ and B₃taken together with the nitrogen atom form a saturated nitrogenheterocycle which contains one or more heteroatoms selected from thegroup consisting of nitrogen, oxygen, and sulfur.
 19. The compound ofclaim 18 wherein:B₁ is selected from the group consisting of methylβ-cyanoethyl or 4-nitrophenylethyl; B₂ and B₃ taken separately areselected from the group consisting of isopropyl, t-butyl, isobutyl andsec-butyl; and B₂ and B₃ taken together is morphollino.
 20. The compoundof claim 17 wherein X and Y taken together is ##STR27## wherein n rangesfrom 2 to
 10. 21. The compound of claim 17 wherein X and Y takentogether is ##STR28## wherein n ranges from 2 to
 10. 22. Aphosphoramidite compound having the formula: ##STR29## wherein: B₁ is aphosphite ester protecting group;B₂, and B₃ taken separately areselected from the group consisting of lower alkyl, lower alkene, aryl,and cycloalkyl containing up to 10 carbon atoms; B₅ is an acid-cleavablehydroxyl protecting group; B is a nucleotide base; and D is the dyecompound of claim 1; wherein when B is purine or 7-deazapurine, thesugar moiety is attached at the N⁹ -position of the purine or7-deazapurine, and when B is pyrimidine, the sugar moiety is attached atthe N¹ -position of the pyrimidine; wherein B and D are linked through alinkage attached to D at one of positions R₁ -R₉ ; and wherein if B is apurine, the linkage is attached to the 8-position of the purine, if B is7-deazapurine, the linkage is attached to the 7-position of the7-deazapurine, and if B is pyrimidine, the linkage is attached to the5-position of the pyrimidine.
 23. The compound of claim 22 wherein thelinkage is ##STR30##
 24. The compound of claim 22 wherein the linkage is25. The compound of claim 22 wherein the linkage is
 26. The compound ofclaim 22 wherein B is selected from the group consisting of uracil,cytosine, deazaadenine, and deazaguanosine.
 27. A labeled nucleotidehaving the formula: wherein:B is a 7-deazapurine, purine, or pyrimidinenucleotide base; W₁ and W₂ taken separately are selected from the groupconsisting of H and OH; W₃ is selected from the group consisting of OH,##STR31## D is the dye compound of claim 1; wherein when B is purine or7-deazapurine, the sugar moiety is attached at the N⁹ -position of thepurine or deazapurine, and when B is pyrimidine, the sugar moiety isattached at the N¹ -position of the pyrimidine; wherein the linkagelinking B and D is attached to D at one of positions R₁ -R₉ ; andwherein if B is a purine, the linkage is attached to the 8-position ofthe purine, if B is 7-deazapurine, the linkage is attached to the7-position of the 7-deazapurine, and if B is pyrimidine, the linkage isattached to the 5-position of the pyrimidine.
 28. The labeled nucleotideof claim 27 wherein B is selected from the group consisting of uracil,cytosine, deazaadenine, and deazaguanosine.
 29. The labeled nucleotideof claim 27 wherein the linkage is ##STR32##30.
 30. The labelednucleotide of claim 27 wherein both W₁ and W₂ are H; and W₃ is
 31. Thelabeled nucleotide of claim 27 wherein W₁ is H; W₂ is OH; and W₃ is 32.A labeled polynucleotide containing a nucleotide having the formula:wherein:B is a 7-deazapurine, purine, or pyrimidine nucleotide base; Z₁is selected from the group consisting of H and OH; Z₂ is selected fromthe group consisting of H, OH, HPO₄, and Nuc, wherein Nuc and thenucleoside are linked by a phosphodiester linkage, the linkage beingattached to the 5'-position of Nuc; Z₃ is selected from the groupconsisting of H, HPO₃, phosphate analogs, and Nuc, wherein Nuc and thenucleoside are linked by a phosphodiester linkage, the linkage beingattached to the 3'-position of Nuc; and, D is a dye compound of claim 1;wherein when B is purine or 7-deazapurine, the sugar moiety is attachedat the N⁹ -position of the purine or deazapurine, and when B ispyrimidine, the sugar moiety is attached at the N¹ -position of thepyrimidine; wherein the linkage linking B and D is attached to D at oneof positions R₁ -R₉ ; and wherein if B is a purine, the linkage isattached to the 8-position of the purine, if B is 7-deazapurine, thelinkage is attached to the 7-position of the 7-deazapurine, and if B ispyrimidine, the linkage is attached to the 5-position of the pyrimidine.33. The labeled polynucleotide of claim 32 wherein B is selected fromthe group consisting of uracil, cytosine, deazaadenine, anddeazaguanosine.
 34. The labeled polynucleotide of claim 32 wherein thelinkage is ##STR33##
 35. A method of polynucleotide sequencingcomprising the steps of: forming a mixture of a first, a second, athird, and a forth class of polynucleotides such that:eachpolynucleotide in the first class includes a 3'-terminaldideoxyadenosine and is labeled with a first dye; each polynucleotide inthe second class includes a 3'-terminal dideoxycytidine and is labeledwith a second dye; each polynucleotide in the third class includes a3'-terminal dideoxyguanosine and is labeled with a third dye; and eachpolynucleotide in the forth class includes a 3'-term dideoxythymidineand is labeled with a forth dye; wherein one of the first, second,third, or forth dyes is the asymmetric benzoxanthene dye of claim 1; theother of the dyes being spectrally resolvable from the asymmetricbenzoxanthene dye and from each other, electrophoretically separatingthe polynucleotides thereby forming bands of similarly sizedpolynucleotides; illuminating the bands with an illumination beamcapable of causing the dyes to fluoresce; and identifying the classes ofthe polynucleotides in the bands by the fluorescence spectrum of thedyes.
 36. The method of claim 35 wherein the other of the dyes areselected from the group consisting of carboxyfluorescein,6-carboxy-4,7,2',7'-tetrachlorofluorescein, and6-carboxy-4,7,2',4',5',7'-hexachlorofluorescein.
 37. A method offragment analysis comprising:forming a labeled polynucleotide fragment,the fragment being labeled with the dye compound of claim 1; subjectingthe labeled polynucleotide fragment to a size-dependent separationprocess; and detecting the labeled polynucleotide fragment subsequent tothe separation process.
 38. The method of claim 37 wherein thesize-dependent separation process is electrophoresis and the detectingis by fluorescence.